Study On The Mechanism Of Lactobacillus Plantarum In Repairing Tight Junction Injury Of Intestinal Epithelial Cells Induced By LPS

Intestinal diseases caused by damage of intestinal epithelial cells have evolved into a global disease,which seriously harms human health.A large number of animal and clinical trials at home and abroad have proved the probiotic effect of lactic acid bacteria in protecting intestinal mucosal barrier,but the mechanism of lactic acid bacteria in promoting intestinal epithelial tight junction is still unclear.Therefore,in this study,Lactobacillus plantarum 1.0318,which can effectively reduce enteritis,was used as the research object to construct a lipopolysaccharide(LPS)-induced tight juncture injury model of intestinal epithelial cells(Caco-2).First,RT-PCR,Western blot and transmission electron microscopy were used to analyze the surface components and metabolites(such as surface proteins,The effects of peptidoglycan and extracellular polysaccharide on the expression of tight junction structure and related proteins;Furthermore,combined with gene overexpression technology,the role of key mi RNAs in the repair of damaged intestinal epithelial tight junctures was investigated.At the same time,Western blotting and immunofluorescence techniques were used to analyze the association between bacterial components regulating MLCK-MLC signaling pathway and repair of tight junction injury.It is expected to reveal the molecular mechanism of probiotic protection of intestinal mucosal barrier from the level of mi RNA and signaling pathway,which has important practical significance for the development of effective probiotics.1.Effect of main components of Lactobacillus plantarum 1.0318 on repair of tight junction injuryL.plantarum 1.0318 adhesin(surface protein,peptidoglycan)and metabolite(exopolysaccharide)were used to interfere the tight junction injury model of Caco-2 cells induced by LPS,respectively.The results showed that the treatment of L.plantarum 1.0318 could effectively improve the resistance value of LPS-induced Caco-2 cells significantly decreased,the permeability of monolayer cells significantly increased,the tight junctions appeared close junctions rupture,the cell gap increased and a large number of intestinal microvilli shed,etc.Compared with peptidoglycan and exopolysaccharide groups,the ultrastructure damage and microvilli shedding of tight junction were less in surface protein group,and the expression l evels of resistance and tight junction proteins ZO-1 and Occludin were higher in surface protein group.Therefore,the surface protein of L.plantarum 1.0318 was used as the experimental object to further explore the mechanism of repair of tight junction in jury.2.Mi RNAs were involved in the repair of tight junction injury by Lactobacillus plantarum1.0318 surface proteinFirstly,the effect of L.plantarum 1.0318 surface protein on the expression of tight protein injury related mi RNAs was observed to determine the key mi RNAs.We found that surface protein intervention reduced LPS-induced mi RNA overexpression,especially significantly inhibited the expression of mi R-200 c.Further studies showed that transfection of mi R-200 c simulant inhibited the ability of surface proteins to upregulate cell resistance and express tight junctions ZO-1,Occludin and Claudin-1,and the simulant transfection eliminated the effect of surface proteins to reduce LPS-induced permeability of Caco-2 cells.These results suggest that L.plantarum 1.0318 surface protein can repair tight protein damage by inhibiting the expression of mi R-200 c in intestinal epithelium.3.L.plantarum 1.0318 surface protein mediated MLCK-MLC pathway repair of tight junction injuryThe LPS-induced tight junction injury model of Caco-2 cells was established,and the effects of surface protein,NF-?B inhibitor and MLCK inhibitor on MLCK-MLC pathway in tight junction injury cells were analyzed.The results showed that NF-?B p65 and MLCK-MLC related proteins were significantly increased in the LPS tight junction injury group,while the expression of tight junction protein was significantly decreased,and the intercellular fluorescence distribution was dispersed,the intensity was weakened,and the intercellular space was in creased After treated with surface protein,NF-?B inhibitor and MLCK inhibitor,MLCK-MLC related pathway protein decreased significantly,and tight junction protein expression increased significantly,tight junction protein fluorescence intensity increased,tight junction distributed along the cell membrane,There was no significant difference among the three groups.In conclusion,L.plantarum 1.0318 surface protein,peptidoglycan and extracellular polysaccharide can effectively repair LPS-induced tight connection injury of Caco-2 cells,and the repair effect of surface protein is significantly better than that of peptidoglycan and extracellular polysaccharide.Further studies found that surface proteins may play a role in the repair of tight junction injury by reducing the expression of mi R-200 c and inhibiting the MLCK-MLC signaling pathway,which provides a new theoretical basis for probiotics to prevent intestinal barrier dysfunction.